Probiotics and fermentation metabolites for the prevention and treatment of disease conditions in animals

ABSTRACT

The present invention is directed to compositions comprising a mixture of microbes and the metabolites produced when the microbes are grown together. The invention is further directed to methods for making and using the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. ProvisionalApplication Ser. No. 62/598,730, filed on Dec. 14, 2017, which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

Not applicable.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates to probiotic microorganisms andfermentation metabolites produced thereby. In particular, the presentdisclosure relates to probiotics and fermentation metabolites that canbe used to treat or prevent a disease or condition in animals.

2. Description of Related Art

Antibiotics have played an integral role in the animal food productionindustry as performance enhancement additives since the discovery ofantibiotics in the early 1940s (McKenna, 2017). In most farms,antibiotics are used to treat clinical infections, control and preventthe spread of disease, and predominantly, to enhance animal growth(American Meat Institute, 2013). Field studies have shown that, whenused as growth enhancers, antibiotics promote flock health bystabilizing the intestinal microbial flora, improving generalperformance and preventing intestinal pathologies (Hassan, 2010). Forexample, it has been shown that the use of tetracycline and tylosines atsub-therapeutic doses over long periods of time can increase the feedconversion ratio in poultry, swine, and cattle (Landers, 2012). Thesebeneficial effects have pushed the industry towards antibiotic-centricproduction practices. Currently, in the United States, twelve classes ofantibiotics may be used at different times in the life cycle oflivestock many of which are analogs of human antibiotics (Landers,2012). In 2012, over 30 million pounds of antibiotics were sold foranimal use, more than four times the amount use for human health,underlying the important role of antibiotics in the food industry(Taillant, 2015). However as the use of antibiotics in food productionhas grown, considerable concerns associated with antibiotics have beenidentified. Notably, the widespread use of antibiotics can lead to thedevelopment of pathogens resistant to antibiotics.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present disclosure is directed to a compositioncomprising at least two microbes and preferably the metabolites producedby the at least two microbes when grown in combination. In certainembodiments, the composition comprises at least two microbes selectedfrom the group consisting of Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium, Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum; and preferably the metabolites produced by the at least twomicrobes when grown in combination.

Another aspect of the present disclosure is directed to a method oftreating or preventing a disease or condition in an animal in needthereof. The method comprises administering to the animal a compositioncomprising at least two microbes and preferably the metabolites producedby the at least two microbes when grown in combination. In certainembodiments, the composition comprises at least two microbes selectedfrom the group consisting of Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium, Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum; and preferably the metabolites produced by the at least twomicrobes when grown in combination. Other aspects and features of thepresent disclosure will be in part apparent and in part pointed outhereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of an example cross-feeding model.

FIG. 2 is a diagram of an example phylogenetic tree.

FIG. 3 is a table summarizing certain experimental results relating tothe production of example compositions.

FIG. 4 is a diagram summarizing results obtained in the performance ofan experiment, notably a heatmap and clustering of kinome profiles. Theraw kinome signal from the peptide array was input into the customsoftware package PIIKA 2. PIIKA 2 combines the biological replicates foreach treatment and tissue, normalizes the data, and generates arepresentative kinome profile. The profiles are compared for relativesimilarity and a heatmap shows the relative phosphorylation of eachpeptide on the array.

FIG. 5 is a diagram summarizing results obtained in the performance ofan experiment, notably a BioSub Heatmap and clustering of treatmentkinome profiles relative to control kinome profiles. The raw kinomesignal from the peptide array was input into the custom software packagePIIKA 2. PIIKA 2 combines the biological replicates for each treatmentand tissue, normalizes the data, and generates a representative kinomeprofile. The profiles for each treatment group are compared to thekinome profile of control groups. The resulting kinome profiles are thencompared for relative similarity and a heatmap shows the relativephosphorylation of each peptide on the array for a given treatment grouprelative to control.

FIG. 6 is an enumeration graph by month.

FIG. 7 presents data for 90° C. stability tests.

FIG. 8 presents for six month stability tests

FIG. 9 presents organic acid profile comparisons of compositions andtheir component strains.

FIG. 10 presents organic acid finger print profiles for batchfermentations.

FIG. 11 presents organic acid finger print profiles for batchfermentations compared between two compositions and their componentstrains.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

Provided herein are methods and compositions for treating and preventinganimal diseases or conditions. The compositions include probiotics,notably a mixture of at least two microbes. In certain embodiments, themicrobes are selected from the group consisting of Lactobacillusreuteri, Pediococcus acidilactici, and Enterococcus faecium. In certainembodiments the composition additionally includes one or more ofPediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillusfermentum and Lactobacillus plantarum. In certain embodiments, thecompositions of the present invention comprise not only microbes, butfermentation products of the microbes when grown in combination, e.g.post-biotic metabolites, notably short chain fatty acids. Other aspectsof the invention are directed to use of the compositions of theinvention treat or prevent animal diseases or conditions.

It has been found that combinations of the microbes of present inventionproduce different fermentation products from the microbes when fermentedalone and/or fermentation products in different ratios from the microbeswhen fermented alone. It has been further found that combinations of themicrobes of the present invention, together with their unique mixture offermentation metabolites, notably the short chain fatty acidmetabolites, offer health benefits to animals. This is beneficialbecause, for example, the feed delivered to production animals is notthe most effective for the animal to be converted into short chain fattyacids by the microbes of the present invention. By delivering a reliableand quantitative amount of the short chain fatty acid metabolites in thecomposition orally, directly to the animal, short chain fatty acids canbe used indirectly in the production of pyruvate, acetyl-coenzyme A, orAcetyl-CoA. These are vital intermediates in the Krebs energy cycle.Further, the compositions of the present disclosure comprise ratios ofshort chain fatty acids that can be delivered in concentrations that arebeneficial to the animal. By contrast, direct oral dosing of short chainfatty acids can limit absorption due to the fact that the amountsdelivered can exceed the amounts an animal's gastrointestinal system isaccustomed to receiving. It is further beneficial that administration ofcompositions of the present invention to an animal may reduce, or eveneliminate, the need for antibiotics.

It has further been found that the compositions and fermentationmetabolites of the present disclosure, such as short chain fatty acids,are heat stable and can tolerate the conditions of high heat duringtransit, storage, and pelletizing all common in the production animalfeed system. The generation of heat stable beneficial fatty acids istherefore a benefit of the present invention in the agriculture industryand other industries.

I. Compositions

In an aspect, a composition of the disclosure comprises at least twomicrobes selected from the group consisting of Lactobacillus reuteri,Pediococcus acidilactici, Enterococcus faecium, Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum; and preferably the metabolites produced by the at least twomicrobes when grown in combination. In certain aspects, the at least twomicrobes are selected from the group consisting of Lactobacillusreuteri, Pediococcus acidilactici, and Enterococcus faecium; preferablytogether with probiotic fermentation products of the microbes when grownin combination, notably short chain fatty acids. In certain embodimentsthe composition additionally includes one or more microbes selected fromthe group consisting of Pediococcus pentosaceus, Lactobacillusacidophilus, Lactobacillus fermentum and Lactobacillus plantarum. Incertain embodiments the compositions of the disclosure comprise at leastone microbe selected from the group consisting of Lactobacillus reuteri,Pediococcus acidilactici, and Enterococcus faecium and at least onemicrobe selected from the group consisting of Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum, preferably together with probiotic fermentation products ofthe microbes when grown in combination, notably short chain fatty acids.

In certain embodiments the composition comprises Lactobacillus reuteri,Pediococcus acidilactici, and Enterococcus faecium; Lactobacillusreuteri, Pediococcus acidilactici, Enterococcus faecium and Pediococcuspentosaceus; Lactobacillus reuteri, Pediococcus acidilactici,Enterococcus faecium and Pediococcus pentosaceus and Lactobacillusfermentum; Lactobacillus reuteri, Pediococcus acidilactici, andLactobacillus acidophilus; or Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium and Lactobacillus acidophilus; ineach case preferably together with probiotic fermentation products ofthe microbes when grown in combination, notably short chain fatty acids.

In certain embodiments, the composition comprises at least two microbesselected from the group consisting of Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium, Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum; preferably together with their metabolites when grown incombination, notably short chain fatty acids. Exemplary compositions ofthe present invention include, but are not limited to compositionscomprising the following; Lactobacillus reuteri and Pediococcusacidilactici; Lactobacillus reuteri and Enterococcus faecium;Lactobacillus reuteri and Pediococcus pentosaceus; Pediococcusacidilactici and Enterococcus faecium; Pediococcus acidilactici andPediococcus pentosaceus; Enterococcus faecium and Pediococcuspentosaceus; Lactobacillus reuteri, Pediococcus acidilactici, andEnterococcus faecium; Lactobacillus reuteri, Pediococcus acidilactici,and Pediococcus pentosaceus; Pediococcus acidilactici, Enterococcusfaecium and Pediococcus pentosaceus; Lactobacillus reuteri andLactobacillus acidophilus; Pediococcus acidilactici and Lactobacillusacidophilus; Enterococcus faecium and Lactobacillus acidophilus;Lactobacillus reuteri, Pediococcus acidilactici, and Lactobacillusacidophilus; and Pediococcus acidilactici, Enterococcus faecium andLactobacillus acidophilus; Lactobacillus reuteri and Lactobacillusacidophilus; Lactobacillus reuteri and Lactobacillus reuteri;Lactobacillus reuteri and Lactobacillus plantarum; Enterococcus faeciumand Lactobacillus acidophilus; Enterococcus faecium and Lactobacillusfermentum; Enterococcus faecium and Lactobacillus plantarum; Pediococcuspentosaceus and Lactobacillus acidophilus; Pediococcus pentosaceus andLactobacillus fermentum; Pediococcus pentosaceus and Lactobacillusplantarum; Pediococcus acidilactici and Lactobacillus acidophilus;Pediococcus acidilactici and Lactobacillus fermentum; Pediococcusacidilactici and Lactobacillus plantarum; Lactobacillus acidophilus andLactobacillus fermentum; Lactobacillus acidophilus and Lactobacillusplantarum; or Lactobacillus fermentum and Lactobacillus plantarum; ineach case alone or in combination with additional microbes selected fromthe group consisting of Lactobacillus reuteri, Pediococcus acidilactici,Enterococcus faecium, Pediococcus pentosaceus, Lactobacillusacidophilus, Lactobacillus fermentum and Lactobacillus plantarum; ineach case preferably together with probiotic fermentation products ofthe microbes when grown in combination, notably short chain fatty acids.

In each of the embodiments discussed above, one or more of the followingstrains may be used: Lactobacillus reuteri PCR7, Pediococcusacidilactici PCLL01, Enterococcus faecium PCEF02, Pediococcuspentosaceus PCPP01, Lactobacillus fermentum PCF01 and a commerciallyavailable Lactobacillus acidophilus PCLA18. Certain embodiments of thepresent invention comprise a combination of Lactobacillus reuteri PCR7,Pediococcus acidilactici PCLL01, Enterococcus faecium PCEF02, andPediococcus pentosaceus PCPP01; or Lactobacillus reuteri PCR7,Pediococcus acidilactici PCLL01, Enterococcus faecium PCEF02,Pediococcus pentosaceus PCPP01 and Lactobacillus fermentum PCF01,preferably together with probiotic fermentation products of the microbeswhen grown in combination. The strain of E. faecium, such as PCEF02,preferably has low toxicity, acceptable for the intended use.

It has been found that combinations of the microbes of the presentinvention produce different fermentation products from the microbes whenfermented alone. In addition, certain of the microbes may be present inreduced numbers after fermentation due to competition among the microbesduring the fermentation process. As detailed in Example 14, acomposition comprising Lactobacillus reuteri, Pediococcus acidilactici,Enterococcus faecium, and Pediococcus pentosaceus produces differentfermentation metabolites from the individual microbes. Othercombinations of microbes producing different fermentation products arelisted in the table below.

TABLE 1 Fermentation Products Acetic Acid Propionic Butryric FormulationKey peaks (ppm) acid (ppm) acid (ppm) Enumeration 17010: PCLA18, 6 peaks2760.05 31.81 20.62 PCLF01, PCLL01 17011 1: PCLA18, 10 peaks  2872.5031.44 3.35 6.5 × 10⁶ PCLF01, PCLL01, PCEF02 17011 2 USDA #1: 8 peaks2336 39.65 3.45 3.9 × 10⁷ PCLA18, PCLL01, PCEF02, PCR7 17001 3: PCLA18,5 peaks 2557.70 34.43 3.8 7.5 × 10⁶ PCLF01, PCLL01, PCR7

In an embodiment, the composition may further comprise one or moreadditional species of microorganisms from the genera selected fromPediococcus; Lactobacillus; Lactococcus; Bifidobacterium; Leuconostoc;Streptococcus; and Bacillus.

In an additional embodiment, the composition may further comprise one ormore of the species of microorganisms selected from the group consistingof Pediococcus pentosaceus; Lactobacillus acidophilus; Lactobacillusplantarum; Lacotobacillus rhamnosus; Lactobacillus fermentum;Lactobacillus bifidus; Lactobacillus brevis; Lactobacillus bulgaricus;Lactobacillus casei; Lactobacillus delbrueckii; Lactobacillus rhamnosus;Lactobacillus helveticus; Lactobacillus johnsonii; Lactobacillus lactis;Lactobacillus lactis ssp. Cremoris; Lactobacillus lactis ssp. Lactis;Lactobacillus paracase; Lactococcus cremoris; Lactococcus lactis;Lactococcus lactis ssp. Cremoris; Bifidobacterium infantis;Bifidobacterium lactis; Bifidobacterium animalis; Bifidobacteriumbifidum, Bifidobacterium longum; Bifidobacterium breve; Leuconostocmesenteroides ssp mesenteroides; Leuconostoc mesenteroides ssp cremoris;Streptococcus bovis; Streptococcus salivarius; Streptococcus salivariusssp. Thermophilus; Bacillus coagulans; Bacillus amyloliquefaciens;Bacillus licheniformis; Bacillus subtilis; and Bacillus lentus.

In each of the embodiments discussed above, one or more of the followingstrains may be used: Lactobacillus reuteri PCR7, Pediococcusacidilactici PCLL01, Enterococcus faecium PCEF02, Pediococcuspentosaceus PCPP01, Lactobacillus fermentum PCF01 and a commerciallyavailable Lactobacillus acidophilus PCLA18.

The microorganisms included in the compositions of the presentdisclosure may be obtained from a collection of microorganisms, such asthe American Type Culture Collection (ATCC) or purchased from commercialvendors of microbial or probiotic strains. In accordance herewith themicroorganisms are then cultured in a fermentation medium under suitableconditions to grow the microorganisms. Certain proprietarymicroorganisms have been deposited with the Agricultural ResearchService Culture Collection NRRL—Northern Research Service CultureCollection Research Laboratory Peroria, Ill., as shown in the followingtable.

TABLE 2 NRRL numbers Pure Cultures # NRRL # Strain # Genius/species 1B-67717 PCLL01 Pediococcus acidilacticia 2 B-67718 PCR7 Lactobacillusreuteri 3 B-67719 PCPP01 Pediococcus pentosaceus 4 B-67720 PCEF02Enterococcus faecium 5 B-67701 PCLA18 Lactobacillus acidophilus

A. Fermentation Process

Many probiotic products on the market simply provide a single microbe orcombination of microbes. However, in certain embodiments of the presentinvention, the compositions of the present invention are produced byfirst fermenting the combinations of microbes in a suitable fermentationmedium to produce metabolites or “postbiotics” that are included in thecomposition.

In an embodiment, for example, a composition comprising at least twomicrobes selected from the group consisting of Lactobacillus reuteri,Pediococcus acidilactici, and Enterococcus faecium, and optionally oneor more of Pediococcus pentosaceus, Lactobacillus acidophilus,Lactobacillus fermentum and Lactobacillus plantarum, including any ofthe compositions described in more detail above, may be added to afermentation media and grown. Further, the at least two microbes mayproduce metabolites that are included in the composition.

Suitable fermentation media may include media comprising, for example,molasses, such as a molasses based on sugar beet juice, sugar canejuice, sucrose, dextrose, glucose, corn steep solids, or corn steepliquor.

In an embodiment, the amount of molasses in the fermentation media maybe from about 1% v/v to about 10% v/v. In some embodiments, the amountof molasses in the fermentation media may be about 1% v/v, about 2% v/v,about 3% v/v, about 4% v/v, about 5% v/v, about 6% v/v, about 7% v/v,about 8% v/v, about 9% v/v, or about 10% v/v.

In some embodiments the fermentation medium may include molasses inhigher amounts. In some embodiments, the fermentation medium may includemolasses:water at a ratio of from about 20:80 v/v to about 35:65 v/v. Inother embodiments, the fermentation medium may include molasses at aratio of about 20:80 v/v, about 25:75 v/v, about 30:70 v/v, or about35:65 v/v.

The fermentation media may also comprise formulation agents, forexample, one or more exogenous prebiotic agents, such asfructooligosaccharide (FOS), a galactooligosaccharide, axylooligosaccharides, an isomaltooligosaccharides, an inulinoligosaccharide, glucooligosaccharides, soybean oligosaccharides,lactosucrose, palatinose, erythritol, or a mannanoligosaccharide toprepare a liquid formulation or a solid formulation, for example about5%.

The fermentation media may also comprise glycerol, for example about0.5% to 3% of the fermentation media.

In certain embodiments the disclosed invention comprises a compositioncomprising at least two microbes selected from the group consisting ofLactobacillus reuteri, Pediococcus acidilactici, and Enterococcusfaecium, and optionally one or more of Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum, including any of the compositions described in more detailabove, added to a fermentation media comprising molasses, and optionallyglycerol, in any of the concentrations above and grown to producemetabolites. In certain embodiments, a composition comprisingLactobacillus reuteri and Pediococcus acidilactici; Lactobacillusreuteri, Pediococcus acidilactici and Lactobacillus acidophilus;Lactobacillus reuteri, Pediococcus acidilactici, and Enterococcusfaecium; Lactobacillus reuteri, Pediococcus acidilactici, Enterococcusfaecium and Pediococcus pentosaceus; Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium and Pediococcus pentosaceus andLactobacillus fermentum; or Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium and Lactobacillus acidophilus isadded to a fermentation media comprising molasses in any of theconcentrations above, and optionally containing glycerol, inulin and/orFOS, and grown to produce metabolites. In certain embodiments, a weakmedia, having low amounts of molasses, and optionally glycerol, is usedwhile still producing a composition with sufficient microbes andmetabolites to treat or prevent a disease or condition. In certainembodiments, one or more of the following strains is be used:Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01,Enterococcus faecium PCEF02, Pediococcus pentosaceus PCPP01,Lactobacillus fermentum PCF01 and a commercially available Lactobacillusacidophilus PCLA18.

The fermentation media may also comprise a nitrogen source, a proteinsource, a trace element, vitamin, a buffering agent, or othercomponents.

In an embodiment, the fermentation media may include a nitrogen source.Suitable nitrogen sources include, without limit, yeast extract,alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine,ammonia citrate, serine, valine, and ammonia sulfate, ammonia salts,whey extract, skim milk powder, corn steep liquor or solids, soya beanmeal, broadbean peptone, corn peptone, pea peptone, wheat peptone,potato peptone, hydrolysate of casein, lupin peptone, malt extract, meatpeptone and rice peptone. In certain embodiments the nitrogen source canbe 1 to 15% of the formulation.

In an embodiment, the fermentation process may include the use of agentssuch as Ammonia hydroxide, sodium hydroxide, sodium carbonate, calciumcarbonate to be used as pH control.

In an embodiment, the fermentation media may include a protein source.Suitable protein sources include, without limit, a soy extract, a yeastextract, a pea extract, broadbean extract, corn extract, a potatoextract, a dairy extract, skim milk power, a tapioca extract, maltextract, meat extract, malt extract and a rice extract. In certainembodiments the protein source can be 1 to 15% of the formulation.

In an embodiment, the fermentation media may include a trace element.Suitable trace elements include, without limit, iron, zinc, copper,manganese, magnesium, molybdenum, and cobalt.

Typical ranges for trace elements are set forth in the following table.

TABLE 3 Trace Elements Component Range (g/L) KH₂PO₄  1.0-4.0 MgSO₄ *7H₂O0.25-3.0 KCL  0.5-12.0 CaCO₃  1.5-17.0 FeSO₄ 0.01-0.1 ZnSO₄  0.1-1.0MnSO₄ 0.01-0.1 CuSO 0.003-0.01 NaMoO₄*2H₂0 0.01-0.1

Table reprinted from Principles of fermentation Technology P. F.Stanbury, A. Whitaker and S. J. Hall second Edition p. 103

In an embodiment, the fermentation media may include a buffering agent.Suitable buffering agents include, without limit, potassium diphosphate,potassium phosphate, sodium carbonate, sodium bicarbonate, potassiumcarbonate, and potassium bicarbonate.

Typical ranges of buffering agents are set forth in the following table.

TABLE 4 Buffering Agents Component Range (g/L) Dipotassium Phosphatedibasic  1-5 Citric acid  0.5-2.5 Monosodium citrate 0.5-10 Disodiumcitrate 0.5-10 Trisodium citrate 0.5-10 Sodium carbonate 0.5-5  Calciumcarbonate 0.5-5  Sodium hydroxide Used to titrate pH into more basicrange Ammonium hydroxide Used to titrate pH into more basic range

In some embodiments, the fermentation medium contains peptone, beefextract, yeast extract, glucose, sodium acetate, polysorbate 80,dipotassium hydrogen phosphate, ammonium acetate, magnesium sulfate, andmanganese sulfate.

In some embodiments, the fermentation medium contains from about 0.5% toabout 2.5% caseine peptone, from about 0.5% to about 3.5% beef extract,from about 0.1% to about 3.5% yeast extract, from about 1.0% to about5.0% glucose, from about 0.1% to about 5.0% sodium acetate, about 0.05%to about 0.5% polysorbate 80, from about 0.05% to about 0.5% dipotassiumhydrogen phosphate, from about 0.05% to about 0.5% ammonium acetate,from about 0.005% to about 0.1% magnesium sulfate, and from about 0.0005to about 0.01% manganese sulfate.

In some embodiments, the fermentation medium may be modified including,for example, by replacing casein peptone with pea peptone, soy peptone,or potato peptone, corn peptone, broadbean peptone, and meat peptone.

In some embodiments, the fermentation medium prior to initiating growthof the microorganisms can comprise the amino acid lysine, which, withoutbeing bound by theory, may enhance the production of the short chainfatty acid butyrate.

In certain embodiments the disclosed invention comprises a compositioncomprising at least two microbes selected from the group consisting ofLactobacillus reuteri, Pediococcus acidilactici, and Enterococcusfaecium, and optionally one or more of Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum, including any of the compositions described in more detailabove, added to a fermentation media comprising molasses in any of theconcentrations above, and optionally glycerol.

Suitable fermentation conditions include a temperature range of fromabout 25° C. to about 42° C. In some embodiments, the fermentationtemperature may be about 25° C., about 26° C., about 27° C., about 28°C., about 29° C., about 30° C., about 31° C., about 32° C., about 33°C., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., about 40° C., about 41° C., or about 42° C.

Typical fermentation times range from 18 to 50 hours. The targetfermentation time is based on a combination of pH, OD reading, totalacidity and alkanlinity of solution.

The vessel in which the microorganisms are cultivated is either held atanaerobic or aerobic conditions.

B. Formulation

In some embodiments, upon conclusion of microbial growth the medium andcells may be formulated into a formulation comprising the probioticmicroorganisms together with fermentation metabolites produced by themicroorganisms, which may include one or more of produced short chainfatty acids, sugars, oligosaccharides, or bacteriocins. In certainembodiments, the formulation comprises secondary and/or tertiarymetabolites. In addition, certain of the microbes may be reduced innumber in the fermentation product due to competition between microbesduring the fermentation process. The formulations may be in liquid orsolid forms.

In an embodiment, the metabolites may include a short chain fatty acid.Suitable short chain fatty acids include, without limit, acetic acid,formic acid, propionic acid, butyric acid, isobutyric acid, valericacid, succinic and isovaleric acid.

The microorganisms during growth produce substantial quantities of shortchain fatty acids in the fermentation medium, for example in the rangeof about 0.5 mg/mL to about 30.0 mg/mL. In some embodiments, the amountof short chain fatty acids produced in the fermentation medium may beabout 0.5 mg/mL, about 1.0 mg/mL, about 1.5 mg/mL, about 2.0 mg/mL,about 2.5 mg/mL, about 3.0 mg/mL, about 3.5 mg/mL, about 4.0 mg/mL,about 4.5 mg/mL, about 5.0 mg/mL, about 5.5 mg/mL, about 6.0 mg/mL,about 6.5 mg/mL, about 7.0 mg/mL, about 7.5 mg/mL, about 8.0 mg/mL,about 8.5 mg/mL, about 9.0 mg/mL, about 9.5 mg/mL, about 10.0 mg/mL,about 10.5 mg/mL, about 11.0 mg/mL, about 11.5 mg/mL, about 12.0 mg/mL,about 12.5 mg/mL, about 13.0 mg/mL, about 13.5 mg/mL, about 14.0 mg/mL,about 14.5 mg/mL, about 15.0 mg/mL, about 15.5 mg/mL, about 16.0 mg/mL,about 16.5 mg/mL, about 17.0 mg/mL, about 17.5 mg/mL, about 18.0 mg/mL,about 18.5 mg/mL, about 19.0 mg/mL, about 19.5 mg/mL, about 20.0 mg/mL,about 20.5 mg/mL, about 21.0 mg/mL, about 21.5 mg/mL, about 22.0 mg/mL,about 22.5 mg/mL, about 23.0 mg/mL, about 23.5 mg/mL, about 24.0 mg/mL,about 24.5 mg/mL, about 25.0 mg/mL, about 25.5 mg/mL, about 26.0 mg/mL,about 26.5 mg/mL, about 27.0 mg/mL, about 27.5 mg/mL, about 28.0 mg/mL,about 28.5 mg/mL, about 29.0 mg/mL, about 29.5 mg/mL, or about 30.0mg/mL.

In another embodiment, the metabolites may include a sugar. Suitablesugars include, without limit, a 4-carbon sugar, a 5-carbon sugar, and a6-carbon sugar. Suitable 4-carbon sugars include, without limit,erythrose and threose. Suitable 5-carbon sugars include, without limit,ribose, arabinose, xylose, and lyxose. Suitable 6-carbon sugars include,without limit, glucose, galactose, mannose, allose, altrose, gulose,idose, and talose. Such metabolites are generally produced asintermediate metabolites that are consumed by the microbes during thefermentation process to produce the final metabolites contained in thecompositions of the present invention.

In another embodiment, the metabolites may include an oligosaccharide.Suitable oligosaccharides include, without limit, fructooligosaccharide,galactooligosaccharide, xylooligosaccharides, erythritol, palatinose,isomaltooligosaccharides, and mannanoligosaccharide. Certainoligosaccharides “prebiotics” are consumed by the bacteria during thefermentaiton process. Some new “prebiotics” are formed by the bacteriaduring the fermentation process.

In still another embodiment, the metabolites may include a bacteriocin.Suitable bacteriocins include, without limit, acidophilin, acidolin, andreutericin.

Yet other fermentation metabolites that can be produced includepeptides, functional proteins, enzymes, amino acids, hydrogen sulfide,hydrogen peroxide, alcohol, carbon dioxide, sulfur dioxide, polyphenols,mannitol, and vitamins.

In some embodiments, upon conclusion of microbial growth the obtainedcompositions, i.e., the growth medium, including the probioticmicroorganisms and fermentation metabolites, may be used directly toprevent or treat a disease or condition in animals. In some embodiments,upon conclusion of growth, the medium and cells may be prepared toobtain a liquid or solid formulation comprising the composition. Thus,for example, the compositions may be included directly into the watersupply of animals.

In an embodiment, the compositions disclosed herein, comprising at leasttwo microbes selected from the group consisting of Lactobacillusreuteri, Pediococcus acidilactici, and Enterococcus faecium, andoptionally one or more of Pediococcus pentosaceus, Lactobacillusacidophilus, Lactobacillus fermentum and Lactobacillus plantarum,including any of the compositions described in more detail above, andthe metabolites produced by the at least two microbes when grown incombination, may be formulated as a liquid or a solid.

Certain embodiments of the invention comprise the compositions disclosedherein, comprising at least two microbes selected from the groupconsisting of Lactobacillus reuteri, Pediococcus acidilactici, andEnterococcus faecium, and optionally one or more of Pediococcuspentosaceus, Lactobacillus acidophilus, Lactobacillus fermentum andLactobacillus plantarum, including any of the compositions described inmore detail above, and the metabolites produced by the at least twomicrobes when grown in combination in a medium comprising molasses, andoptionally glycerol, inulin and/or FOS as described above.

In certain embodiments, a composition comprises Lactobacillus reuteriand Pediococcus acidilactici Lactobacillus reuteri, Pediococcusacidilactici, and Enterococcus faecium; Lactobacillus reuteri,Pediococcus acidilactici, Enterococcus faecium and Pediococcuspentosaceus; or Lactobacillus reuteri, Pediococcus acidilactici,Enterococcus faecium and Lactobacillus acidophilus and the metabolitesproduced by the microbes when grown together in a medium comprisingmolasses in any of the concentrations above, and optionally glycerol,inulin and/or FOS.

It has been found that combinations of the microbes of the presentinvention, together with their unique mixture of fermentationmetabolites, notably the short chain fatty acid metabolites, offerhealth benefits to animals, as discussed in more detail in Examples12-14.

Formulation as a liquid or solid may be achieved by techniques generallyknown in the art. Such techniques for forming solid formulations includespray-drying, evaporation, centrifugation, tangential flow filtration,and microfiltration. Drying equipment that may be used include, withoutlimit, a spray dryer, a convection oven, microwave dryer, Buflowvac,freeze drying, or a rotary drum dryer. Powdered formulations may beprepared to include a bulking agent such as maltodextrin, corn starch,tapioca starch, tapioca dextrose, microcrystalline cellulose, riceflour, rice starch, a prebiotic such as inulin and fos.

In some embodiments, the liquid composition may be concentrated to forma more concentrated liquid composition. The liquid compositions may beconcentrated using techniques generally known in the art. In someembodiments, more concentrated liquid composition may be prepared bycentrifugation, ultra filtration, and evaporation.

In some embodiments, the solid compositions may be powdered orpelletized. The solid compositions may be powdered or pelletized usingtechniques generally known in the art.

In some embodiments, a formulation can be prepared to obtain a specificmicrobial count. In some embodiments, the formulation may contain alactic acid bacterial count of at least about 1.0×105 cfu/gram orcfu/ml. In other embodiments, the formulation may contain a lactic acidbacterial count of at least 1.0×107 cfu/gram or cfu/ml. Typical lacticacid bacterial counts range from 1.0×105 to 1.0×1010 cfu/gram or cfu/ml.

II. Methods

In an aspect, a method of the disclosure comprises treating orpreventing a disease or condition in an animal in need thereof, themethod comprising administering to the animal a composition comprisingat least two microbes selected from the group consisting ofLactobacillus reuteri, Pediococcus acidilactici, and Enterococcusfaecium, and optionally one or more of Pediococcus pentosaceus,Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillusplantarum, including any of the microbe compositions described in moredetail above, together with the metabolites produced by the at least twomicrobes grown together in any of the media above, including any of thecompositions described in more detail above.

In certain embodiments, methods of the disclosure comprise treating orpreventing a disease or condition in an animal in need thereof, themethod comprising administering to the animal a composition thatcomprises Lactobacillus reuteri and Pediococcus acidilacticiLactobacillus reuteri, Pediococcus acidilactici, and Enterococcusfaecium; Lactobacillus reuteri, Pediococcus acidilactici, Enterococcusfaecium and Pediococcus pentosaceus; or Lactobacillus reuteri,Pediococcus acidilactici, Enterococcus faecium and Lactobacillusacidophilus and the metabolites produced by the microbes when growntogether in any of the media described above.

In an embodiment, the disease or condition may be a gastrointestinaldisease. In a further embodiment, the disease or condition may affect aportion of the gastrointestinal tract. In an embodiment, the disease orcondition may affect the duodenal portion of the gastrointestinal tract.In another embodiment, the disease or condition may affect the jejunalportion of the gastrointestinal tract.

In an embodiment, the disease or condition may be necrotic enteritis,Salmonella enteritis, coccidiosis, mastitis, Avian influenza, Fowl pox,infectious bronchitis, Quail bronchitis, Larygotracheitis, NewcastleDisease, mycotoxin poisoning, Porcine Sarcoptic Mange, Pleuropneumonia,gastric ulcers, intestinal torsion, Glasser's Disease, porcineparvovirus, vomiting and wasting disease, porcine epidemic diarrhea,bovine respiratory disease complex, Clostridial disease, bovinerespiratory syncytial, bovine viral diarrhea, Haemophilus somnus,infectious bovine Rhinotracheitis, Parinflenza type 3, PasteurellaHaemolytical, or Pasteurella Multocida. In a further embodiment, thedisease or condition may be necrotic enteritis, Salmonella enteritis, orcoccidiosis mastitis.

In an embodiment, the disease or condition may be a fertility disease orcondition. For example, administration of a formulation of the inventionto an animal may increase the egg laying frequency of poultry, mayreduce the incidence of stillborn or mummy births in mammal productionanimals and/or may increase the number of live births in mammalproduction animals.

In an embodiment, the disease or condition may be caused by aninfectious microbial agent. In some embodiments, the infectiousmicrobial agent may be a Clostridium species. The Clostridium speciesmay be, without limit, Clostridium absonum, Clostridium aceticum,Clostridium acetireducens, Clostridium acetobutylicum, Clostridiumacidisoli, Clostridium aciditolerans, Clostridium acidurici, Clostridiumaerotolerans, Clostridium aestuarii, Clostridium akagii, Clostridiumaldenense, Clostridium aldrichii, Clostridium algidicarnis, Clostridiumalgidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum,Clostridium alkalicellulosi, Clostridium amazonense, Clostridiumaminophilum, Clostridium aminovalericum, Clostridium amygdalinum,Clostridium amylolyticum, Clostridium arbusti, Clostridium arcticum,Clostridium argentinense, Clostridium asparagiforme, Clostridiumaurantibutyricum, Clostridium autoethanogenum, Clostridium baratii,Clostridium barkeri, Clostridium bartlettii, Clostridium beijerinckii,Clostridium bifermentans, Clostridium bolteae, Clostridium bornimense,Clostridium botulinum, Clostridium bowmanii, Clostridium bryantii,Clostridium butyricum, Clostridium cadaveris, Clostridium caenicola,Clostridium caminithermale, Clostridium carboxidivorans, Clostridiumcarnis, Clostridium cavendishii, Clostridium celatum, Clostridiumcelerecrescens, Clostridium cellobioparum, Clostridiumcellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,Clostridium cellulovorans, Clostridium chartatabidum, Clostridiumchauvoei, Clostridium chromiireducens, Clostridium citroniae,Clostridium clariflavum, Clostridium clostridioforme, Clostridiumcoccoides, Clostridium cochlearium, Clostridium colletant, Clostridiumcocleatum, Clostridium colicanis, Clostridium colinum, Clostridiumcollagenovorans, Clostridium cylindrosporum, Clostridium difficile,Clostridium diolis, Clostridium disporicum, Clostridium drakei,Clostridium durum, Clostridium estertheticum, Clostridium estertheticumestertheticum, Clostridium estertheticum laramiense, Clostridium fallax,Clostridium felsineum, Clostridium fervidum, Clostridium fimetarium,Clostridium formicaceticum, Clostridium frigidicarnis, Clostridiumfrigoris, Clostridium ganghwense, Clostridium gasigenes, Clostridiumghonii, Clostridium glycolicum, Clostridium glycyrrhizinilyticum,Clostridium grantii, Clostridium haemolyticum, Clostridium halophilum,Clostridium hastiforme, Clostridium hathewayi, Clostridium herbivorans,Clostridium hiranonis, Clostridium histolyticum, Clostridiumhomopropionicum, Clostridium huakuii, Clostridium hungatei, Clostridiumhydrogeniformans, Clostridium hydroxybenzoicum, Clostridium hylemonae,Clostridium jeddahense, Clostridium jejuense, Clostridium indolis,Clostridium innocuum, Clostridium intestinale, Clostridium irregulare,Clostridium isatidis, Clostridium josui, Clostridium kluyveri,Clostridium lactatifermentans, Clostridium lacusfryxellense, Clostridiumlaramiense, Clostridium lavalense, Clostridium lentocellum, Clostridiumlentoputrescens, Clostridium leptum, Clostridium limosum, Clostridiumlitorale, Clostridium liquoris, Clostridium lituseburense, Clostridiumljungdahlii, Clostridium lortetii, Clostridium lundense, Clostridiumluticellarii, Clostridium magnum, Clostridium malenominatum, Clostridiummangenotii, Clostridium mayombei, Clostridium maximum, Clostridiummethoxybenzovorans, Clostridium methylpentosum, Clostridium moniliforme,Clostridium neopropionicum, Clostridium nexile, Clostridiumnitrophenolicum, Clostridium novyi, Clostridium oceanicum, Clostridiumorbiscindens, Clostridium oroticum, Clostridium oryzae, Clostridiumoxalicum, Clostridium papyrosolvens, Clostridium paradoxum, Clostridiumparaperfringens (Alias: C. welchii), Clostridium paraputrificum,Clostridium pascui, Clostridium pasteurianum, Clostridium peptidivorans,Clostridium perenne, Clostridium perfringens, Clostridium pfennigii,Clostridium phytofermentans, Clostridium piliforme, Clostridiumpolysaccharolyticum, Clostridium polyendosporum, Clostridium populeti,Clostridium propionicum, Clostridium proteoclasticum, Clostridiumproteolyticum, Clostridium psychrophilum, Clostridium puniceum,Clostridium punense, Clostridium purinilyti cum, Clostridiumputrefaciens, Clostridium putrificum, Clostridium quercicolum,Clostridium quinii, Clostridium ramosum, Clostridium rectum, Clostridiumroseum, Clostridium saccharobutylicum, Clostridium saccharogumia,Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum,Clostridium sardiniense, Clostridium sartagoforme, Clostridiumsaudiense, Clostridium senegalense, Clostridium scatologenes,Clostridium schirmacherense, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium sporosphaeroides, Clostridiumstercorarium, Clostridium stercorarium leptospartum, Clostridiumstercorarium stercorarium, Clostridium stercorarium thermolacticum,Clostridium sticklandii, Clostridium stramini solvens, Clostridiumsubterminale, Clostridium sufflavum, Clostridium sulfidigenes,Clostridium swellfunianum, Clostridium symbiosum, Clostridium tagluense,Clostridium tarantellae, Clostridium tepidiprofundi, Clostridiumtermitidis, Clostridium tertium, Clostridium tetani, Clostridiumtetanomorphum, Clostridium thermaceticum, Clostridiumthermautotrophicum, Clostridium thermoalcaliphilum, Clostridiumthermobutyricum, Clostridium thermocellum, Clostridium thermocopriae,Clostridium thermohydrosulfuricum, Clostridium thermolacticum,Clostridium thermopalmarium, Clostridium thermopapyrolyticum,Clostridium thermosaccharolyticum, Clostridium thermosuccinogenes,Clostridium thermosulfurigenes, Clostridium thiosulfatireducens,Clostridium tyrobutyricum, Clostridium uliginosum, Clostridiumultunense, Clostridium ventriculi, Clostridium villosum, Clostridiumvincentii, Clostridium viride, Clostridium vulturis, Clostridiumxylanolyticum, or Clostridium xylanovorans. In a further embodiment, theClostridium species may be Clostridium perfringens.

In an embodiment, the composition may reduce the amount of pathogenspresent in the animal's feces.

In an embodiment, the composition may reduce the ammonia content of theanimal's feces.

In an embodiment, the composition may reduce the amount of methaneproduced and expelled by the animal either in respiratory action or indigestive gas release to the environment.

In an embodiment, the composition may effect the feed conversion ratepositively.

In an embodiment, the composition may reduce animal mortality rate.

In an embodiment, the composition may increase the average daily weightgain.

In an embodiment, the composition may increase egg shell quality and eggweight.

In an embodiment, the composition may modulate the gastrointestinalmicrobiome of an animal. In another embodiment, the composition maymodulate the gastrointestinal immune response of an animal. In otherembodiments, the composition may modulate the phosphorylation ofgastrointestinal proteins of an animal.

In some embodiments, the solid compositions may be added to a feedration of the animal or provided to the animal as a supplement.

In an embodiment, the composition may be provided in a solid form andadded to an animal feed ration. In some embodiments, the solidcomposition may be added to an animal feed ration in an amount of fromabout 0.1 kg/ton to about 2.0 kg/ton of feed. In other embodiments, thesolid composition may be added to an animal feed ration in an amount ofabout 0.1 kg/ton, about 0.25 kg/ton, about 0.5 kg/ton, about 0.75kg/ton, about 1.0 kg/ton, about 1.25 kg/ton, about 1.5 kg/ton, about1.75 kg/ton, or about 2.0 kg/ton.

In some embodiments, the liquid compositions may be added to thedrinking water supply of the animal.

In an embodiment, the composition may be provided in a liquid form andadded to an animal's drinking water. In some embodiments, the liquidcomposition may be added to animal's drinking water in an amount of fromabout 0.1 mL/day/animal to about 10 mL/day/animal. In other embodiments,the liquid composition may be added to an animal's drinking water in anamount of about 0.1 mL/day, about 0.25 mL/day, about 0.5 mL/day, about1.0 mL/day, about 1.25 mL/day, about 1.5 mL/day, about 1.75 mL/day,about 2.0 mL/day, about 2.25 mL/day, about 2.5 mL/day, about 2.75mL/day, about 3.0 mL/day, about 3.25 mL/day, about 3.5 mL/day, about3.75 mL/day, about 4.0 mL/day, about 4.25 mL/day, about 4.5 mL/day,about 4.75 mL/day, about 5.0 mL/day, about 6.25 mL/day, about 6.5mL/day, about 6.75 mL/day, about 7.0 mL/day, about 7.25 mL/day, about7.5 mL/day, about 7.75 mL/day, about 8.0 mL/day, about 8.25 mL/day,about 8.5 mL/day, about 8.75 mL/day, about 9.0 mL/day, about 9.25mL/day, about 9.5 mL/day, about 9.75 mL/day, or about 10.0 mL/day.

In an embodiment, the composition may be provided in a liquid form andadded to a chicken's drinking water. In some embodiments, the liquidcomposition may be added to a chicken's drinking water in an amount offrom about 0.1 mL/day to about 2 mL/day/animal. In other embodiments,the liquid composition may be added to a chicken's drinking water in anamount of about 0.1 mL/day/animal, about 0.25 mL/day/animal, about 0.5mL/day/animal, about 1.0 mL/day/animal, about 1.25 mL/day/animal, about1.5 mL/day/animal, about 1.75 mL/day/animal, or about 2.0 mL/day/animal.

In an embodiment, the composition may be provided in a liquid form andadded to a pig's drinking water. In some embodiments, the liquidcomposition may be added to a pig's drinking water in an amount of fromabout 0.5 mL/day/animal to about 10 mL/day/animal. In other embodiments,the liquid composition may be added to a pig's drinking water in anamount of about 0.5 mL/day·animal, about 0.75 mL/day/animal, about 1.0mL/day/animal, about 1.25 mL/day/animal, about 1.5 mL/day/animal, about1.75 mL/day/animal, about 2.0 mL/day/animal, about 2.25 mL/day/animal,about 2.5 mL/day/animal, about 2.75 mL/day/animal, about 3.0,mL/day/animal, about 3.25 mL/day/animal, about 3.5 mL/day/animal, about3.75 mL/day/animal, about 4.0 mL/day/animal, about 4.25 mL/day/animal,about 4.5 mL/day/animal, about 4.75 mL/day/animal, about 5.0mL/day/animal, about 5.25 mL/day/animal, about 5.5 mL/day/animal, about5.75 mL/day/animal, about 6.0 mL/day/animal, about 6.25 mL/day/animal,about 6.5 mL/day/animal, about 6.75 mL/day/animal, about 7.0mL/day/animal, about 7.25 mL/day/animal, about 7.5 mL/day/animal, about7.75 mL/day/animal, about 8.0 mL/day/animal, about 8.25 mL/day/animal,about 8.5 mL/day/animal, about 8.75 mL/day/animal, about 9.0mL/day/animal, 9.25 mL/day/animal, about 9.50 mL/day/animal, about 9.75mL/day/animal or about 10.0 mL/day/animal

Those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Definitions

Bacterial species are presented herein by Latin names in accordance withthe Linnaean taxonomic biological classification system. Accordingly,reference is made to microorganisms which can be with identified withreference to certain genus, species, subspecies and strain names.

The term “microbiome” as used herein, refers to the community ofmicrobial species present in the gastrointestinal system of an animal.

The terms “probiotic” and “probiotic microorganism” as used herein,refers to a composition of one or more species of microorganisms whichwhen orally administered can provide health benefits to an animal.

The term “prebiotic” as used herein, means a non-microbial ingredientfor optional inclusion in a probiotic formulation capable of inducinggrowth or activity of probiotic microorganisms in the gastrointestinalsystem.

The term “postbiotic” as used herein, means a non-viable bacterialproducts or metabolic by products from probiotic microorganisms thathave biologic activity in the host.

The terms “short chain fatty acids” or “SCFAs”, as may beinterchangeably used herein, include acetic acid, formic acid, propionicacid, butyric acid, isobutyric acid, valeric acid, succinic acid, andisovaleric acid.

As used herein, “animal” refers to, but are not limited to, a human, alivestock animal, a companion animal, a lab animal, and a zoologicalanimal. In another embodiment, the animal may be a livestock animal. Inan embodiment, the animal may be a bovine animal, a porcine animal, or apoultry animal. In other embodiments, the animal may be a cow, a pig, ora chicken. Non-limiting examples of suitable livestock animals mayinclude pigs, cows, horses, goats, sheep, llamas, and alpacas. In yetanother embodiment, the subject may be a companion animal. Non-limitingexamples of companion animals may include pets such as dogs, cats,rabbits, and birds. In yet another embodiment, the animal may be azoological animal. In an alternative embodiment, the animal may be ahuman.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Method of Procurement of Microorganisms for Use in aFermentation Cocktail Formulation

The strains used were single strain organism cultures stored in 15% to25% glycerol:media. The strains were unique species obtained by theinventors from Pure Cultures, Inc., or obtained by bio prospecting fromcommercial dietary supplements or obtained from the USDA/NRRL culturebank.

Example 2 Method of Specimen Collection and Isolation of Bacteria fromFeces

Fresh feces samples were gathered from healthy canine in Colorado, USAand immediately frozen at approximately −18° C. Fecal matter was addedto peptone water (10% wt./vol.) and vortexed to fully homogenizesamples. Serial dilutions of the fecal homogenate were then streakedonto MRS agar for isolation. Single colonies were selected, furtherpurified via MRS agar isolation, and subsequently enriched in MRS brothonce a pure colony was obtained. Enriched media was then diluted with30% glycerol:water solution. 1 ml Aliquotes were then placed in 2 mlcryo vials and stored at −80° C.

Example 3 Method of Specimen Collection and Isolation of Bacteria fromCommercial Dietary Supplement Product

Purchased probiotic product was inoculated into to non-animal MRS media.Inoculated media was incubated at 37° for approximately 15 hours. Asterile inoculation loop was dipped into the media with growth andstreaked on to a non-animal MRS agar plate. Plate was incubatedovernight approximately 18 hours. Single colony was picked and placed inautoclaved non-animal media and incubated at 37° C. Enriched media wasthen diluted 1:1 with 50% glycerol:water solution. 1 ml Aliquotes werethen placed in 2 ml cryo vials and stored at −80° C.

Example 4 Method of 16sRNA Sequencing for Identification of Strains

Strains were sent to MIDI Laboratories Newark, DE 19713. Approximately500 base pairs were sequenced and compared to D16M3 Library Revision3.01.

Example 5 Method of Full Genomic Sequencing of Bacterial Strains

Each strain was processed according to established SOPs at the ATCC-CTM.Genomic DNA was extracted from each strain using a QIAmp DNA Mini Kit(Qiagen Cat No./ID 51304) and subsequently quantified fluorometricallyusing a QUBIT 2.0 fluorometer as per the manufacturer's instructions(ThermoFisher Scientific).

The workflow for whole genome sequencing was conducted as recommended byIllumina. Library preparation was performed using the Nextera XT DNALibrary Prep Kit (Illumina Cat No. FC-131-1096) as per manufacturer'sinstructions. Once the library was prepared and quality controlled—itwas then sequenced using an Illumina MiSeq with the MiSeq Reagent Kit V3600 cycles (Illumina Cat No. MS-102-3003). The resulting sequences wereevaluated for read quality and accuracy using the application softwareFASTQC from Babrahm

Bioinformatics, Babraham Institute, Cambridge, UK(<https://www.biinformatics.babraham.ac.uk/projects/fastqc/>). The QC'd,filtered sequences were then assembled into contigs and annotated usingATCC-CTM's proprietary Advanced Microbial Genomics (AMG) Platform.

Example 6 Method for Assaying SCFAs

A HPLC method using Agilent 1100 series HPLC unit. A Bio-Rad OrganicAcid Analysis Column Aminex HPX-87H Ion Exclusion Column with 0.2 NH2SO4 stationary mobile phase. Heated to 60° C.

Diet based cross-feeding mechanism, as shown in FIG. 1, is a key featurein the mammalian, avian and reptilian gut microbiome. In order tounderstand the cross feeding mechanism, further modeling was conductedand compared to the whole genome metabolic models (of the same fourstrains) against several key SCFAs producers (reference gut microbiomespecies). Along with our four whole genome metabolic models the finalcross feeding model was constructed using Bacteroides_thetaiotaomicron,Escherichia_coli_str_K_12, Clostridium_ramosum_VPI,Lactobacillus_plantarum. Cross feeding model was run on two key SCFAs(e.g. butyrate) producing metabolites i.e. lactate (secondary) and xylan(primary). Interestingly, all four strains showed significant crossfeeding interactions with other reference strains. This resulthighlights the fact that the strains have the genetic/metabolicpotential to efficiently produce the SCFAs using primary (xylan) andsecondary (lactate) metabolic precursors for SCFAs production.

Example 7 Method for Evaluating Pathogen Inhibition

Antimicrobial activities of the LAB strains were tested with welldiffusion assay using TSB agar plates. Known pathogen standard wasinoculated overnight in TSB and incubated 37° C. for 24 hours+/−4 hours.100 μL were spread on the TSB agar plate. Holes were punched in the agarand 25 μL of overnight ferment broth was inoculated into the well andallowed to incubate overnight at 37° C. The size of the inhibition halois measured in mL. The results for certain strains are shown in thefollowing table.

TABLE 5 E. coli inhibition of strains with different media Welldiffusion Strain media agar Lab notebook ref. (mm) Lactobacillus A TSBSKK1016p.006 14 acidophilus PCLA18 Lactobacillus reuteri A TSBSKK1016p.006 11 R4 Lactobacillus reuteri A TSB SKK1016p006 18 PCR7 PCR7Lactobacillus reuteri A TSB SKK1016p.006 12 LR2 commercially availablestrain Lactobacillus B TSB SKK1016p.006 15 acidophilus PCLA18Lactobacillus reuteri B TSB SKK1016p.006 15 PCR4 Lactobacillus reuteri BTSB SKK1016p.006 18 PCR7 Lactobacillus reuteri B TSB SKK1016p.006 15 LR2commercially available strain

Example 8 Method for BLASTing Sequence Data Against Known Databases

As shown in FIG. 3, draft genomes were annotated (Pathway and Enzymelevel) using Prokka pipeline (<https://github.com/tseemann/prokka>).Taxonomic status was assigned to each genome using 400 marker genedataset and finally compared against the reference genomes. A maximumlikelihood tree was constructed (bootstrap=1000) with all four draftgenomes and their top 50 closest phylogenetic neighbors.

Whole genome based phylogenetic analysis (above mentioned) clearlyestablished the species level identity for each genome. The strain levelidentity of each genome was analyzed using pairwise whole genome averagenucleotide identity (ANI) analysis with an established strain levelcut-off of 99.9%. Pairwise ANI calculations clearly highlighted that allfour strains i.e. L. reuteri PCR7, P. acidilactici PCLL01, E. faeciumPCEF02, and L. fermentum PCF1 are novel strains in comparison to theirclosest species level genotype.

Using simulated micobiome data (from customized genome dataset of SCFAproducing genomes) In situ replication rates were predicted across eachgenome using iRep tool (<https://github.com/christophertbrown/iRep>).Downstream analysis of in situ replication rates clearly highlighted thefast in situ growth in a gut environment (simulated using >500 culturedreference species genomes). Biopieces were used to extract the nearcomplete sequences of the protein coding genes for mucus- andfibronectin-binding factors, implicated in adhesion to intestinal cells.Interestingly, L. fermentum PCF1 and L. reuteri PCR7 showed positive hit(BLASTx, e-value=10-e5) for mucin and fibronectin-binding proteins.Using Abricate (<https://github.com/tseemann/abricate>) pipeline, thedraft genome assemblies were further screened for any ARG genes presentin the close proximity of transposons and/or Integrons. As expected fora probiotic strain, none of the four draft genome showed any ARG gene inthe close proximity of a transposons and/or Integron. These resultsclearly highlight the minimal horizontal gene transfer potential of thestrains. Virulence factors were annotated by searching the SubsystemFeature Counts of the RAST output for those factors identified in theVirulence, Disease and Defense subsystem. The presence of bacteriocinsin the assembled sequences was determined by comprehensive searches ofthe BACTIBASE database (<http://bactibase.hammamilab.org/main.php>).BACTIBASE contains calculated or predicted physicochemical properties of230 bacteriocins produced by both Gram-positive (206) and Gram-negativebacteria (19). The information in this database allows rapid predictionof structure/function relationships in addition to the target organismsof these peptides, which provides better exploitation of theirbiological activity in both the medical and food sectors. Each assemblywas used individually as queries for the searches.

EXAMPLE 9 Method for Evaluation of SCFAs with Gas Chromatography and aFID Detector

Sample extracts were analyzed on an Agilent 6890 Series GasChromatograph equipped with flame ionization detection (GC-FID; AgilentInc., Santa Clara, Calif.). Injection rate was 10:1 split ratio, andinlet temperature was 22° C. and translet line temperature was held at230° C. Separation was achieved on a 30 m TG-WAX-A column(ThermoScientific, 0.25 mm ID, 0.25 um film thickness) at 100° C. for 1minute and ramp rate of 8° C. per minute to 180° C., held at 180° C. for1 minute, ramped to 200° C. at 20° C./min and held for 5 minutes. Heliumcarrier flow was maintained at 1.2 mL per minute. Short chain fattyacids were quantified using 5-point standard curves of commerciallypurchased standards (Sigma, St. Louis, Mo., USA) and normalized tointernal standard signal. As shown in FIG. 4, acetic acid has aretention time of 5.4 and 5.5 minutes. Propionic acid has a retentiontime of 6.5 minutes. Butyric acid has a retention time of 7.7 minutes.Lactic acid has a retention time of 13.2 and 14.78 minutes, whereasascorbic acid has retention times of 14.04 and 15.09 minutes.

Example 10 Method for Evaluation of Genome Assemblies Using ProkkaPipeline

Raw genomic sequences were assembled into contents using IDBA assembler,a novo assembler for single-cell and metagenomic sequencing. Contigswere annotated using UniProt database. Annotated results were parsedusing BioPython GenBank. Using custom BASH and Python scripts theannotated files were parsed into enzyme and pathway level functionalmatrices, as shown in the table below.

TABLE 6 Heat Stabilized proteins Name of Heat Stable Protein PCLF1PCEF02 PCLL01 PCR7 18 kDa heat shock protein 0 1 0 0 Heat shock protein15 1 1 0 1 Heat-inducible transcription 1 1 1 1 repressor HrcA Chaperoneprotein CipB 0 1 1 0 Chaperone protein DnaJ 1 2 1 1 Chaperone proteinDnaK 1 1 1 1 Molecular chaperone Hsp31 and 0 2 1 0 glyoxalase Tablelists the number of copies present in the genome.

Example 11 Probiotic Formulation

Strains of the following microorganisms were obtained: Lactobacillusacidophilus PCLA18, Lactobacillus reuteri, strain PCR7, Pediococcusacidilacti, strain PCLL01, and Enterococcus faecium PCEF02 were combinedand grown in a liquid growth medium comprising approximately 5%molasses, 5% inulin and 2.5% v/v glycerol. Upon completion of growth themedium together with the bacteria was harvested. The harvested materialcan be used as a probiotic.

Example 12 Phosphorylation of Duodenal and Jejunal Proteins by aProbiotic Formulation

A probiotic formulation was prepared as described in Example 11 and usedin chicken feeding experiments. The duodenal and jejunal portions of thedigestive tract kinome (Manning et al., 2002) of the chickens wereevaluated. The following groups of chickens were evaluated: untreatedcontrol (group (i)); chickens treated the probiotic formulation (group(ii)); chickens challenged with Clostridium perfringens (group (iii));chickens challenged with Clostridium perfringens and treated with theprobiotic formulation (group (iv)); chickens treated with a Coccidiosisvaccine and probiotic formulation (group (v)); and chickens withClostridium perfringens and treated with a Coccidiosis vaccine andprobiotic formulation (group (vi)). Biological replicate samples from 5birds per group were combined to generate representative kinomeprofiles. Following data combination and normalization, cluster analysiswas performed on the resulting 12 kinome profiles (one for each tissue(jejunum and duodenum) (2) X treatment group ((i)-(vi)) (6)) using thecustom software package PIIKA 2 (Trost et al., 2013). FIG. 6 shows aheatmap and clustering displaying the relative similarity between the 12profiles based on phosphorylation of individual proteins. A further heatmap was created representing the kinome profiles of the treatment/tissuecombinations relative to the control kinome profiles for each respectivetissue, as show in FIG. 7.

An effect of the probiotic formulation can be seen in FIG. 6. Jejunumtissue obtained from chickens treated with the probiotic formulationtend to cluster together. Duodenum tissue samples do not seem to developa particular clustering pattern. Jejunum tissue samples obtained fromchickens not treated with the probiotic formulation also form a cluster.This suggests that the probiotic treatment has a distinct effect onjejunal tissue, separate from the effect of the Clostridium perfringenschallenge

Referring to FIG. 7, duodenum tissue samples again shows littledifferentiation between groups. Jejunum tissue samples again seem tocluster based on metabolite treatment with the non-probiotic formulationtreated group standing separate. The most removed from the metabolitecluster is the Clostridium perfringens induced necrotic enteritisprobiotic treated group, suggesting the greatest changes occurring inthis group relative to control when compared to the other metabolitetreated groups.

Example 13 Identification of Jejunal Biological Processes and SignalingPathways Following Treatment with a Probiotic Formulation

In a peptide array obtained from the groups of chickens (i)-(iv)described in Example 12, individual peptides exhibiting differentialphosphorylation patterns in jejunal tissue samples, relative tountreated control chickens (i) were identified. These individual weresubjected to a protein STRING analysis (Szklarczyk D et al., 2015;<https://string-db.org>) to reveal relevant biological processes (GO),and a KEGG analysis (Kanesha M, Goto S, 2000;<https://www.genome.jp/kegg/pathway.html>) to reveal relevant signalingpathways. Table 7 and Table 8 show the STRING and KEGG results obtainedusing probiotic treated chickens (group (ii)); Table 9 and Table 10 showthe STRING and KEGG results obtained using Clostridium perfringenschallenged chickens (group (iii)); and Table 11 and Table 12 show theSTRING and KEGG results obtained using Clostridium perfringenschallenged chickens treated with probiotic (group (iv)).

It is noted that some of the biological processes may overlap betweenthe Tables, the members of the biological processes had to be unique inorder to be included in the analysis. Therefore, the same terms mayappear between groups, but they are being enriched in different waysbetween the groups. This indicates that the various biological processesenriched are behaving in different ways between the treatment groups.

Highlighted in Table 7 and Table 9 are the terms related to immunesignaling. All of the biological processes highlighted in the probioticgroups (Table 7) also appear in the Clostridium perfringens challengegroups (Table 9), but it is important to note that the lists used togenerate these tables were unique to each group. Thus, although the sameterms appear, they represent changes in the same biological processesbetween the treatment groups. Interestingly, the Probiotic+Clostridiumperfringens challenge group (Table 11) does not have the same immunerelated biological pathways enriched in the top 20 pathways from theSTRING analysis. This shows that the overlap of the two treatments,probiotic and Clostridium perfringens challenge, likely are impartingtheir own unique effects in jejunal tissue, which, when broughttogether, do not enrich new immune related biological processes.

With respect to Table 8, Table 10, and Table 12, it is noted that theKEGG pathways may overlap between the Tables, the members of thepathways had to be unique in order to be included in the analysis,therefore, the same terms may appear between groups, but they are beingenriched in different ways between the groups. This indicates that thevarious KEGG pathways enriched are behaving in different ways betweenthe treatment groups.

TABLE 7 Top 20 Biological Processes (GO) enriched uniquely in jejunumtissue of probiotic treated chickens compared to untreated chickensfalse # of discovery Biological Process proteins rate cellular responseto chemical stimulus 36 8.62E−12 cellular response to organic substance32 3.00E−11 phosphorylation 25 9.43E−11 transmembrane receptor proteintyrosine kinase 21 1.13E−10 signaling pathway enzyme linked receptorprotein signaling pathway 23 2.10E−10 protein phosphorylation 211.03E−09 regulation of signaling 34 7.35E−09 regulation of response tostimulus 36 6.90E−08 regulation of immune response 19 6.90E−08phosphorus metabolic process 27 7.12E−08 response to organic substance31 7.12E−08 regulation of cell communication 33 9.58E−08 regulation ofimmune system process 23 1.57E−07 phosphate-containing compoundmetabolic process 26 1.94E−07 cell surface receptor signaling pathway 276.22E−07 regulation of signal transduction 29 6.58E−07 response tochemical 36 9.23E−07 positive regulation of immune system process 172.70E−06 innate immune response 18 2.70E−06 single-organism metabolicprocess 37 4.38E−06

TABLE 8 Top 20 KEGG pathways enriched uniquely in jejunum tissue ofprobiotic treated chickens compared to untreated chickens false # ofdiscovery KEGG Pathway proteins rate PI3K-Akt signaling pathway 146.90E−09 Fc gamma R-mediated phagocytosis 9 6.90E−09 Epstein-Barr virusinfection 11 1.56E−08 Pancreatic cancer 7 2.58E−07 Non-alcoholic fattyliver disease (NAFLD) 9 3.08E−07 Ras signaling pathway 10 4.99E−07Toxoplasmosis 8 4.99E−07 Influenza A 9 5.57E−07 Osteoclastdifferentiation 8 7.90E−07 Hepatitis C 8 9.40E−07 Measles 8 9.40E−07Pathways in cancer 11 9.70E−07 Hepatitis B 8 1.42E−06 VEGF signalingpathway 6 2.15E−06 Toll-like receptor signaling pathway 7 2.40E−06 TNFsignaling pathway 7 3.14E−06 Fc epsilon RI signaling pathway 6 3.49E−06Adipocytokine signaling pathway 6 3.61E−06 Prolactin signaling pathway 64.08E−06 Adherens junction 6 4.19E−06

TABLE 9 Top 20 Biological Processes (GO) enriched uniquely in jejunumtissue of Cp challenged chickens compared to unchallenged chickens false# of discovery Biological Process proteins rate regulation of immuneresponse 20 3.90E−09 protein metabolic process 38 1.15E−08 cellularprotein metabolic process 35 1.31E−08 positive regulation of metabolicprocess 35 1.80E−08 regulation of phosphate metabolic process 241.80E−08 intracellular signal transduction 26 1.80E−08 defense response23 2.02E−08 regulation of phosphorylation 22 2.02E−08 positiveregulation of immune response 16 2.02E−08 immune response-regulatingsignaling 15 2.44E−08 pathway regulation of protein modification process24 2.47E−08 protein phosphorylation 18 3.17E−08 innate immune response19 3.93E−08 regulation of immune system process 22 4.69E−08 activationof immune response 14 5.11E−08 response to nitrogen compound 18 8.11E−08immune response-activating signal 13 1.35E−07 transduction Fc receptorsignaling pathway 11 1.35E−07 Fc-epsilon receptor signaling pathway 101.46E−07 regulation of protein phosphorylation 20 1.60E−07

TABLE 10 Top 20 KEGG pathways enriched uniquely in jejunum tissue of Cpchallenged chickens compared to unchallenged chickens false # ofdiscovery KEGG Pathway peptides rate Insulin signaling pathway 84.18E−06 T cell receptor signaling pathway 7 5.66E−06 Neurotrophinsignaling pathway 7 1.14E−05 Natural killer cell mediated cytotoxicity 71.36E−05 Focal adhesion 8 2.00E−05 Prostate cancer 6 2.00E−05 Estrogensignaling pathway 6 2.93E−05 Pathways in cancer 9 4.47E−05 Glioma 56.17E−05 Fc epsilon RI signaling pathway 5 8.24E−05 Osteoclastdifferentiation 6 8.79E−05 Prolactin signaling pathway 5 8.79E−05Hepatitis C 6 0.000102 ErbB signaling pathway 5 0.000183 MAPK signalingpathway 7 0.000416 Endometrial cancer 4 0.000436 Viral carcinogenesis 60.000532 Non-small cell lung cancer 4 0.000532 FoxO signaling pathway 50.000803 Measles 5 0.00108

TABLE 11 Top 20 Biological Processes (GO) enriched uniquely in jejunumtissue of Cp challenged chickens treated with probiotic compared tonon-challenged untreated chickens false # of discovery BiologicalProcess proteins rate protein autophosphorylation 11 9.87E−09 enzymelinked receptor protein signaling pathway 17 5.74E−08 transmembranereceptor protein tyrosine kinase 15 1.05E−07 signaling pathwayregulation of cellular protein metabolic process 21 1.08E−05 regulationof intracellular signal transduction 17 1.08E−05 positive regulation ofkinase activity 11 1.18E−05 positive regulation of lipid metabolicprocess 7 1.18E−05 intracellular signal transduction 18 4.55E−05regulation of protein kinase activity 12 4.55E−05 response to externalstimulus 18 4.88E−05 positive regulation of catalytic activity 164.88E−05 regulation of multicellular organismal process 20 4.88E−05positive regulation of molecular function 17 6.06E−05 peptidyl-tyrosinephosphorylation 7 6.85E−05 protein phosphorylation 12 0.000123regulation of cellular component biogenesis 11 0.000123 regulation ofprotein modification process 16 0.000124 regulation of proteinphosphorylation 14 0.000128 positive regulation of intracellular signal12 0.000132 transduction axon guidance 9 0.000151

TABLE 12 Top 20 KEGG pathways enriched uniquely in jejunum tissue of Cpchallenged chickens treated with probiotic compared to non-challengeduntreated chickens false # of discovery KEGG Pathway proteins rate AMPKsignaling pathway 7 4.40E−06 Acute myeloid leukemia 5 3.23E−05 Chemokinesignaling pathway 6 0.000441 Insulin signaling pathway 5 0.00125PI3K-Akt signaling pathway 6 0.00685 Leukocyte transendothelialmigration 4 0.00685 Pathways in cancer 6 0.00685 mTOR signaling pathway3 0.0146 ErbB signaling pathway 3 0.0329 Endocytosis 4 0.0329 Fc gammaR-mediated phagocytosis 3 0.0329 Regulation of actin cytoskeleton 40.0397 Proteoglycans in cancer 4 0.0405 Thyroid cancer 2 0.0405

Example 14 Evaluation of Animal Health Parameters Following Treatmentwith a Probiotic Formulation

A probiotic formulation was prepared as described in Example 11 and usedin chicken feeding experiments. Various animal health parameters wereevaluated. The following groups of chickens were evaluated: untreatedcontrol (T1); chickens treated the probiotic formulation (T2); chickenschallenged with a coccidiosis vaccine (T3); chickens challenged withClostridium perfringens and treated with a coccidiosis vaccine (T4);chickens challenged with Clostridium perfringens (T5); chickenschallenged with Clostridium perfringens and treated with probioticformulation (T6); chickens treated a coccidiosis vaccine and withprobiotic formulation (T7); and chickens challenged with Clostridiumperfringens and treated with a coccidiosis vaccine and a probioticformulation (T8). The following health parameters were evaluated: (a)colony forming units of Clostridium perfringens in the intestine; (b)body weight gain; (c) Clostridium perfringens induced necrotic enteritislesions; and (d) mortality. The experimental design is furthersummarized in Table 13.

TABLE 13 Experimental design Body Coccidial Clostridium Lesion TreatmentGroups Weights Challenge perfringens Score T1* Negative Control Day 1,14, 21 No No Day 21 T2* Neg control + Probiotic Day 1, 14, 21 No No Day21 T3* Cocci Vac Day 1, 14, 21 Day 14 No Day 21 T4 Cocci Vac +Clostridium Day 1, 14, 21 Day 14 Day 17, 18, and 19* Day 21 perfringensT5 Positive Clostridium Day 1, 14, 21 No Day 17, 18, and 19* Day 21perfringens control T6 Clostridium perfringens + Day 1, 14, 21 No Day17, 18, and 19* Day 21 Probiotic T7* Cocci Vac + Probiotic Day 1, 14, 21Day 14 No Day 21 T8 CocciVac + Clostridium Day 1, 14, 21 No Day 17, 18,and 19* Day 21 perfringens + Probiotic The following results wereobtained when comparing T8 with T4 and T5: (a) A significant reductionin total CFU of Clostridium perfringens in the intestine was observed(1.7 × 105 (T8) vs 6.2 × 105 (T4) and 14.0 × 105 (T5), respectively);(b) A significant larger body weight gain: (781 gram (T8) vs 697 gram(T4) and 695 gram (T5), respectively). As a comparison, T1 (negativecontrol) had an average weight gain of 805 gram; (c) A significantreduction in lesion score: (0.56(T8) vs 2.3 (T4) and 3.0 (T5),respectively); and (d) A significant reduction in mortality: (1/50 (T8)vs 12/50 (T4) and 21/50 (T5), respectively).

Example 15 Producing and Detecting a Unique Blend of Organic Acids in aConsortium Liquid Cocktail for Poultry Health

Introduction. Lactic acid bacteria (Lactobacilli) have been studied fordecades, and one well-known function of these organisms is their abilityto metabolize short-chain organic acids from 5- and 6-carbon sugarsources (Urdaneta, et al., 1995). Examples of such acids include acetic,butyric, formic, lactic, and propionic (Zalán, Hudáček , Štětina,Chumchalová, & Halász, 2009).

Methods. All techniques were performed aseptically. For eachfermentation trial, up to four individual Lactobacilli strains werechosen from 15%-25% glycerol/MRS freezer stocks. They were subsequentlyincubated for 1 to 2 days at to 37° C.+/−2° C. Following this initialpropagation, all of the strains were combined in pasteurized media anddeionized water and incubated for 5 to 15 days at 30 to 37° C.+/−2° C.The resulting samples were individually resolved by HPLC, utilizing AOAC986.13 (mod). Analysis was performed with a refractive index detector,and peaks were identified by comparison to organic acid standards.

Results. Small organic acids are the end products of the fermentation ofa chosen carbon (sugar) source by a chosen combination of Lactobacilli.The selection of a particular carbon source and microorganismcombination may result in the production of three main organic acids, asis displayed in Table 14 and Table 15.

TABLE 14 Organic acid production Single Organism in Major Peaks notLactic acid and acetic molasses media (lactic and acetic acid) acidPCLL01 3, 4, 5 area ratios 3:1:2 Lactic acid 7,000 ppm PediococcusAcetic acid 1,000 ppm acidilactici PCEF02 3, 5 no peak 4 ratio 1:5Lactic acid 10,000 ppm Enterococcus Acetic acid 3,000 ppm faecium PCR7Peak 3 and 5 No peak Lactic acid 7,000 ppm Lactobacillus 4. Huge peak at20.93 Acetic acid 2,000 ppm reuteri retention time PCPP01 3, 4, 5 arearatios 3:1:2 Lactic acid 9,000 ppm Pediococus Acetic acid <1,000 ppmpentosaceus

Multiple batches have been evaluated using the Pure Cultures in-houseSOP SCFAHLC001 for purity and short chain fatty acid finger print. Thefirst batch 17014 containing strains Lactobacillus reuteri PCR7,Pediococcus acidilactici PCLL01, Pediococcus pentosaceus PPPC01, andEnterococcus faecium PCEF01, produced for trial #1 at Texas A&MUniversity was assayed after six months and in addition it was heated to90° C. for 5 minutes. A second batch 18007, containing strainsLactobacillus fermentum PCLF01, Lactobacillus reuteri PCR7, Pediococcusacidilactici PCLL01, Pediococcus pentosaceus PCPP01, Enterococcusfaecium PCEF02 was assayed using the same evaluation SOP for purity.

TABLE 15 Organic acid production Consortium product with 4 Organisms inMajor Peaks not Lactic acid and acetic molasses media (lactic and aceticacid) acid 17014 3, 4, 5 area ratios 1:1:1 Lactic acid 3,000 ppmOriginal assay Acetic acid <1,000 ppm 17014 heated to 90° C. 3, 4, 5area ratios 1:1:1 Lactic acid 3,000 ppm Acetic acid <1,000 ppm 17014 sixmonth 3, 4, 5 area ratios 1:1:1 Lactic acid 4,000 ppm check Acetic acid1,000 ppm 18007 3, 4, 5 area ratios 1:1:1 Lactic acid 4,000 ppm Aceticacid 2,000 ppm

Discussion. The techniques described herein provide a quick and reliablemethod for the detection and initial characterization of organic acidsproduced in solution by fermentation. Providing evidence of areproducible fermentation process and the ability to produce a uniqueand proprietary formulation. The careful selection of carbon sourcesand/or microorganisms can result in a controlled fermentationenvironment for the production of specific organic acids. As discussedin the Introduction, the selection of particular organic acids isimportant for both the weight-gain and anti-pathogen inhibition inpoultry.

Example 16 Enumeration Tests

Based on USP<2021> Microbial Enumeration Tests for Dietary Supplements.Total Microbial Count plate method.

Samples are collected and shipped to third party laboratory. Samples areshipped overnight with ice packs to ensure stored <10° C.

Media Preparation:

Lactobacilli MRS agar+0.5 g/L-Cysteine is prepared per directions onpackage with the addition of L Cysteine. MRS media+0.5 g/L-Cysteine isprepared per directions on package with the addition of L Cysteine.

10.0 g of sample is aseptically weighed and diluted in 90 ml sterilebuffered peptone. Sample and buffer are homogenized. 1 ml is removed anddiluted with 99 ml peptone buffer to make −3 log dilution. Continue toserial dilute until 3 dilutions are made around the estimate of thefinal lactic acid bacteria count.

Inoculate 0.1 ml per plate of MRS agar+Cysteine. Spread with sterilespreader. Allow inoculum to absorb into the agar. Place in anaerobechamber and incubate at 37° C. for 48 to 72 hours. After appropriateincubation time colonies are counted on agar plates with a target yieldbetween 30-300 per plate. Results are shown in FIG. 6 and Table 16.

TABLE 16 Lactic acid bacteria enumeration of batches 7.5 months 8.3months Batch Date of Original @ room @ room Final Product # manufactureLAB assay temperature temperature specification 10M cfu/ml 18001 Feb. 1,2018 430000000 300,000 20000 2.0 months ≥5.0 × 10{circumflex over ( )}7cfu/ml cfu/ml cfu/ml 3.3 months ≥1.0 × 10{circumflex over ( )}7 3.9months 5.4 months Batch Date of Original @ room @ room Final Product #manufacture LAB assay temperature temperature specification 10M cfu/ml18004 Mar. 29, 2018 272,000,000 220,000,000 1,100,000 4.0 months ≥5.0 ×10{circumflex over ( )}7 cfu/ml cfu/ml cfu/ml 4.5 months ≥1.0 ×10{circumflex over ( )}7 3.9 months 5.4 months Batch Date of Original @room @ room Final Product # manufacture LAB assay temperaturetemperature specification 10M cfu/ml 18006 May 19, 2018 245,000,0001,200,000 200,000 1.5 months ≥5.0 × 10{circumflex over ( )}7 cfu/mlcfu/ml cfu/ml 2.5 months ≥1.0 × 10{circumflex over ( )}7 1.23 months 4.1months Batch Date of Original @ room @ room Final Product # manufactureLAB assay temperature temperature specification 10M cfu/ml 18007 Aug.25, 2018 420,000,000 81,000,000 13,000,000 2.2 months ≥5.0 ×10{circumflex over ( )}7 cfu/ml cfu/ml cfu/ml 4.0 months ≥1.0 ×10{circumflex over ( )}7 1.23 months Date of Original @ room FinalProduct Batch # manufacture LAB assay temperature specification 10Mcfu/ml 181023FV Oct. 25, 2018 1,100,000,000 190,000,000 1.8 months ≥5.0× 10{circumflex over ( )}7 cfu/ml cfu/ml 2.6 months ≥1.0 × 10{circumflexover ( )}7 Average 2.2 months @ ≥5.0 × 10{circumflex over ( )}7; Average4.83 months @ ≥1.0 × 10{circumflex over ( )}7

Batch 18001 contains strains Lactobacillus fermentum PCLF01,Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01, Pediococcuspentosaceus PCPP01, Enterococcus faecium PCEF01

Batch 18004 contains strains Lactobacillus fermentum PCLF01,Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01, Pediococcuspentosaceus PCPP01, Enterococcus faecium PCEF01

Batch 18006 contains strains Lactobacillus fermentum PCLF01,Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01, Pediococcuspentosaceus PCPP01, Enterococcus faecium PCEF01

Batch 18007 contains strains Lactobacillus fermentum PCLF01,Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01, Pediococcuspentosaceus PCPP01, Enterococcus faecium PCEF01

Batch 181023 contains strains Lactobacillus reuteri PCR7, Pediococcusacidilactici PCLL01, Pediococcus pentosaceus PCPP01, Enterococcusfaecium PCEF01

Example 17 90° C. Stability Test.

Formulation batch 17014 containing strains Lactobacillus reuteri PCR7,Pediococcus acidilactici PCLL01, Pediococcus pentosaceus PPPC01, andEnterococcus faecium PCEF01 in a molasses media was placed in a heatingoven that at a temperature set point of 90° C. Approximately 10 mls ofsample were heated to 90° C. in heating oven. A piece of aluminum foilwas placed on top of the beaker while it was being heated. Once thesample reached 90° C. a timer was started. At 5 minutes sample wasremoved from the oven and placed in a refrigerator until temperature wasapproximately 40° C. Sample was sent to Midi Laboratories in Omaha, Neb.where Organic Acids were assayed using AOAC 986.13 (mod) assay.

A HPLC method using Agilent 1100 series HPLC unit. A Bio-Rad OrganicAcid Analysis Column Aminex HPX-87H Ion Exclusion Column with 0.2 NH2SO4 stationary mobile phase.

Organic acid chromatography was evaluated for similarities in peaks andareas of each peak present between a sample that was not heated and asample that was heated to 90° C. Step 1. Determine that there are 9peaks present in both chromatographs. If there is a new peak present ineither sample ensure that its area is not >1.5% of all areas addedtogether. Step 2. Ensure that the area of peaks 3,4 and 5 are in theratio of 1:1:1. Step 3. Verify that the lactic acid and acetic acidconcentrations in both samples are +/−0.5% of the concentration eachsample. Results are shown in FIG. 7.

Example 18 Six Month Stability Test

Formulation batch 17014 containing strains Lactobacillus reuteri PCR7,Lactobacillus fermentum PCLF01, Pediococcus acidilactici PCLL01,Pediococcus pentosaceus PPPC01, and Enterococcus faecium PCEF01 in amolasses media was stored at room temperature approximately 22° C.+/−2°C. It was stored in an 8 ounce plastic HDPE bottle. After six months ofstorage a sample was sent to Midi Laboratories in Omaha, Neb. whereOrganic Acids were assayed using AOAC 986.13 (mod) assay.

A HPLC method using Agilent 1100 series HPLC unit. A Bio-Rad OrganicAcid Analysis Column Aminex HPX-87H Ion Exclusion Column with 0.2 NH2SO4 stationary mobile phase.

Organic acid chromatography was evaluated for similarities in peaks andareas of each peak present between a sample that was evaluated 5 daysafter the batch was made against a sample that was six months old andstored at room temperature. Step 1. Determine that there are 9 peakspresent in both chromatographs. If there is a new peak present in sixmonth sample ensure that its area is not >1.5% of all areas addedtogether. Step 2. Ensure that the area of peaks 3,4 and 5 are in theratio of 1:1:1. Step 3. Verify that the lactic acid and acetic acidconcentrations in six month sample are +/−0.5% of the concentration ofthe original sample. Results are shown in FIG. 8.

Example 19 Drying Stability Tests

Formulation 17003 containing strains Lactobacillus reuteri PCR7,Lactobacillus fermentum PCLF01, and Enterococcus faecium PCEF01 in amolasses media was sent to Bluegrass Dairy for spray drying and rollerdrum drying. Using techniques standard to the art of drying. It wasnecessary to add nearly 85% w/w maltodextrin for both drying processes.

After drying process samples were sent to Midi Laboratories in Omaha,Neb. where Organic Acids were assayed using AOAC 986.13 (mod) assay.

A HPLC method using Agilent 1100 series HPLC unit. A Bio-Rad OrganicAcid Analysis Column Aminex HPX-87H Ion Exclusion Column with 0.2 NH2SO4 stationary mobile phase.

Organic acid chromatography was evaluated for similarities in peaks andareas of each peak present between a sample that was the liquid startingmaterial against dried powders from both the spray drying and rollerdrum processes. Step 1. Determine that the concentration of lactic acidin the dried material is within the theoretical concentration +/−25%.Calculation: Take the lactic acid result in % and multiply by 99 anddivide by 85 (the % of dilution with carrier). If Acetic acid is <0.5%in the original liquid it may not be present in dried material as itmore susceptible to heat degradation and volatizing when heated.

Example 20 Comparison of Batches and Component Strains

Organic acid profiles for certain multi strain batches were determinedusing the methods described above with respect to Example 18.Comparisons between 4 compositions and their components are shown inFIG. 10. FIG. 11 depicts an organic acid “fingerprint” profile for fourcompositions. FIG. 12 depicts a comparison of certain compositions andcomponent strains. ET8 contains strains Lactobacillus reuteri PCR7,Pediococcus acidilactici PCLL01, Pediococcus pentosaceus PCPP01,Enterococcus faecium PCEF02. ET4 contains strains_ Lactobacillus reuteriPCR7, Pediococcus acidilactici PCLL01, Pediococcus pentosaceus PCPP01,Enterococcus faecium PCEF02.

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Zalán, Z., Hudáček, J., Štětina, J., Chumchalová, J., & Halász, A.(2009). Production of organic acids by Lactobacillus strains in threedifferent media. European Food Research and Technology, 395-404.

All cited references are herein expressly incorporated by reference intheir entirety for their relevant disclosure.

From the foregoing it will be seen that this invention is one welladapted to attain all ends and objectives herein-above set forth,together with the other advantages which are obvious and which areinherent to the invention.

Since many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that allmatters herein set forth OPT or shown in the accompanying drawings areto be interpreted as illustrative, and not in a limiting sense.

While specific embodiments have been shown and discussed for purposes ofillustration, various modifications may of course be made, and theinvention is not limited to the specific forms or arrangement of partsand steps described herein, except insofar as such limitations areincluded in the following claims. Further, it will be understood thatcertain features and subcombinations are of utility and may be employedwithout reference to other features and subcombinations. This iscontemplated by and is within the scope of the claims.

What is claimed and desired to be secured by Letters Patent is asfollows:
 1. A composition comprising Lactobacillus reuteri, Pediococcusacidilactici, Enterococcus faecium, and Pediococcus pentosaceus, and themetabolites produced by the microbes grown in combination.
 2. Thecomposition of claim 1 further comprising Lactobacillus fermentum, andthe metabolites produced by the at least two microbes grown incombination.
 3. The composition of claim 1, wherein the compositioncomprises Lactobacillus reuteri PCR7, Pediococcus acidilactici PCLL01,Enterococcus faecium PCEF02, and Pediococcus pentosaceus PCPP01.
 4. Thecomposition of claim 3, wherein the composition further comprisesLactobacillus fermentum PCF01.
 5. The composition any of claim 1,wherein the metabolites comprise a short chain fatty acid selected fromthe group consisting of acetic acid, formic acid, propionic acid,butyric acid, isobutyric acid, valeric acid, isovaleric acid, succinicacid, and combinations thereof.
 6. The composition of claim 1, whereinthe metabolites comprise a sugar selected from the group consisting of a4-carbon sugar, a 5-carbon sugar, a 6-carbon sugar, and combinationsthereof.
 7. The composition any claim 1, wherein the metabolitescomprise an oligosaccharide.
 8. The composition of claim 7, wherein theoligosaccharide is selected from the group consisting of afructooligosaccharide, a galactooligosaccharide, a xylooligosaccharides,an isomaltooligosaccharides, an inulin oligosaccharide, amannanoligosacchSeearide, and combinations thereof.
 9. The compositionof claim 1, wherein the metabolites comprise a bacteriocin.
 10. Thecomposition of claim 9, wherein the bacteriocin is selected from thegroup consisting of acidophilin, acidolin or reutericin.
 11. Thecomposition of claim 1, wherein the metabolites comprise metabolitesproduced when the microbes are grown in a medium comprising molasses.12. The composition of claim 12, wherein the medium additionallycomprises glycerol and a prebiotic selected from an inulin and fos. 13.The composition of claim 1, wherein a portion of the microbes comprisingthe composition are viable after storage at room temperature for 2.2months >50,000,000 cfu/ml, preferably for 4.8 months >10,000,000 cfu/ml.14. The composition of claim 1, wherein an organic acid profile of thecomposition is stable after storage for six months at room temperature.15. The composition of claim 1, wherein an organic acid profile of thecomposition is stable when the composition is heated to 90 C for 6minutes.
 16. The composition of claim 1, wherein an organic acid profileof the composition is stable after roller drying and/or spray drying thecomposition
 17. A method of treating or preventing a disease orcondition in an animal in need thereof, the method comprisingadministering to the animal the composition of claim
 1. 18. The methodof claim 17, wherein the disease or condition is a gastrointestinaldisease.
 19. The method of claim 17, wherein the disease or condition isnecrotic enteritis, Salmonella enteritis, or coccidiosis mastitis. 20.The method of claim 17, wherein the disease or condition is caused by aninfectious microbial agent.
 21. The method of claim 20, wherein thedisease or condition is caused by a Clostridium species.
 22. The methodof claim 17, wherein the composition modulates the gastrointestinalmicrobiome of the animal.
 23. The method of claim 17, wherein thecomposition modulates the gastrointestinal immune response of theanimal.
 24. The method of claim 17, wherein the composition modulatesthe phosphorylation of gastrointestinal proteins of the animal.
 25. Themethod of claim 17 wherein the composition is provided in a solid form,and included in an animal feed ration at a concentration of from about0.25 kg/ton to about 2.0 kg/ton of feed.
 26. The method of claim 17,wherein the composition is formulated into a liquid form and added tothe drinking water system of the animal.
 27. The method of claim 17,wherein the composition is dosed from about 0.4 mL/animal day to about5.0 mL/animal/day.
 28. The method of claim 17, wherein the compositionis formulated to contain at least a lactic acid bacterial count of about1.0×10⁵.
 29. A composition produced by the method of fermenting thecomposition of claim 1 in a media comprising molasses.
 30. Thecomposition of claim 29, wherein the media further comprises glyceroland a prebiotic selected from the group consisting of an inulin and fos.